New derivatives of echinocandine, their preparation process and their use as antifungals

ABSTRACT

A subject of the invention, in all possible isomer forms as well as their mixtures, is the compounds of formula (I):  
                 
in which 
     either R 1 : H or CH 3  and    R 2 : cyclohexyl substituted by an amine, a (CH 2 )b—C≡N radical or R 1  and R 2  together with the nitrogen which carries-them form a ring with 3, 4 or 5 carbons optionally substituted by an amine    R 3 : hydrogen, methyl or hydroxyl    R 4 : hydrogen or hydroxyl R: represents a linear,. branched or cyclic chain    T: hydrogen, methyl, CH 2 CONH 2 , CH 2 C≡N, a (CH 2 ) 2 NH 2  or (CH 2 ) 2 Nalk + X −  radical, X halogen and alk alkyl    Y: hydrogen, hydroxyl, halogen or OSO 3 H, W: H or OH,    Z: H, CH 3 . The compounds of formula (I) have antifungal properties.

PRIOR APPLICATIONS

This application is a division of U.S. patent application Ser. No.10/018,073 filed Dec. 4, 2001, now U.S. Pat. No. ______ which is a 371of PCT/FR00//01568 filed Jun. 8, 2000.

The present invention relates to new derivatives of echinocandine, theirpreparation process and their use as antifungals.

A subject of the invention is, in all possible isomer forms as well astheir mixtures, the compounds of formula (I):

in which

-   either R₁ represents a hydrogen atom or a methyl radical.-   R₂ represents a cyclohexyl radical substituted by an amine, a    CH₂CH₂NHCH3 radical, a CH₂CHCH₃NH₂ radical, a    radical, a CHCH₃CH₂NH₂ radical, a —(CH₂)aOH radical, a representing    an integer comprised between 1 and 8, a (CH₂)b—C≡N radical, b    representing an integer comprised between 1 and 8, a CHCH₃C₆H₅    radical, a (CH₂)—C(CH₃)₂NHCOCF₃ radical, a CHCH₃(CH₂)dOH radical, d    representing an integer comprised between 1 and 8-   or R₁ and R₂ form together with the nitrogen which carries them a    ring with 3, 4 or 5 carbons optionally substituted by an amine-   R₃ represents a hydrogen atom, a methyl or hydraxyl radical-   R₄ represents a hydrogen atom or a hydroxyl radical-   R represents a linear or branched or cyclic chain containing up to    30 carbon atoms, optionally containing one or more heteroatoms, one    or more heterocycles or a linear, branched or cyclic acyl radical    containing up to 30 carbon atoms optionally containing one or more    heteroatoms and/or one or more heterocycles,-   T represents a hydrogen atom, a methyl radical, a CH₂CONH₂ radical,    CH₂C≡N, a (CH₂)₂NH₂ or (CH₂)₂Nalk⁺X⁻ radical, X being a halogen atom    and alk an alkyl radical containing up to 8 carbon atoms,-   Y represents a hydrogen atom, a hydroxyl radical or a halogen atom    or an OSO₃H. radical or one of the salts of this radical,-   W represents a hydrogen atom or an OH radical,-   Z represents a hydrogen atom or a methyl radical, as well as the    addition salts with acids of the products of formula (I).

Among the addition salts with acids, there can be mentioned those formedwith mineral acids, such as hydrochloric, hydrobromic, sulphuric orphosphoric acid or with organic acids such as formic, acetic,trifluoroacetic, propionic, benzoic, maleic, fumaric, succinic,tartaric, citric, oxalic, glyoxylic and aspartic acids, alkanesulphonicacids, such as methane or ethane sulphonic acid, arylsulphonic acidssuch as benzene or paratoluene sulphonic acids.

Among the preferred compounds of the invention, there can quiteparticularly be mentioned the compounds of formula I in which Trepresents a hydrogen atom, those in which W represents a hydrogen atom,those in which Z represents a methyl radical, those in which Yrepresents a hydrogen atom, those in which R₃ represents a methylradical, those in which R₄ represents a hydroxyl radical, and those inwhich R represents a

radical.

A most particular subject of the invention is the compounds of formula Iin which R represents a

chain or a

chain.

Among the preferred compounds of the invention, there can be quiteparticularly mentioned the compounds of formula I in which R₁ is ahydrogen atom, those in which R₂ is a

radical,those in which R2 is a

radical, a

radical or a

radical or also those in which R₂ is a

radical

A most particular subject of the invention is the compounds of formula(I), the preparation of which is given hereafter in the experimentalpart and in particular the products of Examples 2 and 3.

The compounds of formula (I) have useful antifungal properties; they arein particular active on Candida albicans and other Candida such asCandida glabrata, krusei, tropicalis, pseudotropicalis, parapsilosis andAspergillus fumigatus, Aspergillus flavus, Cryptococcus neoformans.

The compounds of formula (I) can be used as medicaments in man oranimals, in particular to combat invasive candidosis in theimmunosuppressed, digestive, urinary, vaginal or cutaneous candidosis,cryptococcosis, for example neuromeningeal, pulmonary or cutaneouscryptococcosis, bronchopulmonary and pulmonary aspergillosis andinvasive aspergillosis in the immunosuppressed.

The compounds of the invention can also be used in the prevention ofmycotic illnesses in the congenital or acquired immunosuppressed.

The compounds of the invention are not limited to a pharmaceutical use,they can also be used as fungicides in fields other than thepharmaceutical field.

Therefore a subject of the invention is, as antifungal compounds, thecompounds of formula (I) as well as their addition salts with acids.

A subject of the invention is also the compounds of formula (I), asmedicaments.

A most particular subject of the invention is the pharmaceuticalcompositions containing as active ingredient at least one compound offormula (I) or one of its addition salts with pharmaceuticallyacceptable acids. These compositions can be administrered by oral,rectal, parenteral route or by local route as a topical application onthe skin and mucous membranes, but the preferred routes are the oral andparenteral routes.

They can be solid or liquid and can be presented in the pharmaceuticalforms commonly used in human medicine, such as for example, plain orsugar-coated tablets, gelatin capsules, granules, suppositories,injectable preparations, ointments, creams, gels; they are preparedaccording to the usual methods. The active ingredient or ingredients canbe incorporated in the excipients usually used in these pharmaceuticalcompositions, such as talc, gum arabic, lactose, starch, magnesiumstearate, cocoa butter, aqueous or non-aqueous vehicles, fatty matter ofanimal or vegetable origin, paraffin derivatives, glycols, variouswetting, dispersing or emulsifying agents, preservatives.

These compositions can also be presented in the form of a powderintended to be dissolved extemporaneously in an appropriate vehicle, forexample apyrogenic sterile water.

The dose administered is variable according to the illness treated, thepatient in question, the administration route and the productconsidered. It can be, for example, comprised between 50 mg and 1 g (perday by oral or parenteral route, in adults for the products of Examples2 and 3.

A subject of the invention is also a preparation process characterizedin that a compound of formula (II)

in which R, R₃, R₄, T, Y, W and Z retain their previous meaning, issubjected to the action of an amine or an amine derivative capable ofintroducing the

radical in which R₁ and R₂ retain their previous meaning and if desiredis subjected to the action of a reducing agent and/or of an aminefunctionalization agent, and/or an acid in order to form the salt of theproduct obtained, and/or a separation agent of the different isomersobtained, and the sought compound of formula (I) is thus obtained.

The compounds of formula (II) described and claimed in the PatentApplication WO 99 29716 can be prepared according to a processcharacterized in that a compound of formula (III)

in which the different substituents retain their previous meaning issubjected to the action of an agent capable of replacing NH₂ with NHR, Rretaining its previous meaning in order to obtain the compound offormula (IV)

which is subjected to the action of trimethylsilyl iodide in order toobtain the corresponding compound of formula (II)

The following examples illustrate the invention without however limitingit.

Preparation 1: “nucleus” of deoxymulundocandine

2 g of deoxymulundocandine is dissolved in 20 ml of DMSO. This solutionis poured into a suspension containing 120 g of Actinoplanes utahensisFH2264 in 870 ml of a KH2PO4, K2HPO4 buffer (pH: 6.8). The reactionmixture is maintained under agitation for 70 hours at 30° C. Filtrationis carried out. The mycelium is washed with the phosphate buffer (pH:6.8). The washing liquids and the filtrate are combined. The productobtained is chromatographed on a DIAION Hp 20 resin and a product isobtained which is used as it is hereafter.

EXAMPLE 1 1-[4-[((2S)-2-amino-2-methylethyl)-amino]N2-[[4′-(octyloxy)[1,1′-biphenyl]-4-yl]-carbonyl]-L-ornithine]-4-[4-(4-hydroxyphenyl)-L-threonine]5-L-serineechinocandin B trifluoroacetate (isomer A and isomer B).

Stage A: 1-[(4R,5R)-4,5-dihydroxy-N2-[[4′-(octyloxy)[1,1′-biphenyl]-4-yl]-carbonyl]-L-ornithine]-4-[4-(4-hydroxy-phenyl)-L-threonine]-5-L-serineechinocandin B.

1-Preparation of the Ester

632 mg of 2,3,4,5,6 pentafluorophenol and 695 mg of N,N′-dicyclohexylcarbodiimide are added to 1 g of4′-octyloxy-[1,1′-biphenyl]4-carboxylic acid in 22 ml oftetrahydrofuran, followed by agitation for 22 hours at ambienttemperature and filtration. The solvents are eliminated under reducedpressure, the residue is taken up in ether, agitated at approximately35° C., followed by filtration, the solvent is evaporated followed bydrying and 1.46 g of expected product is recovered, which is used as itis.

2- Coupling

677 mg of the deoxymulundocandine “nucleus” obtained in Preparation 1 isintroduced into 16 ml of DMF. The solution obtained is agitated for 5minutes and 793 mg of pentafluorophenyl4′-(octyloxy)-[1,1′-biphenyl]-4-carboxylate obtained above is added. Thereaction mixture is maintained under agitation and a nitrogen atmospherefor 24 hours. The reaction mixture is filtered and concentrated. Theresidue is taken up in ether, triturated, maintained under agitation for25 minutes, separated, washed with ethyl ether, chromatographed onsilica while eluting with a mixture of methylene chloride, methanol,water (86/13/1) then (80/20/1). The sought product is thus obtained.Yield 73%.

Stage B:1-[N2-[[4′-(octyloxy)-[1,1′-biphenyl]-4-yl]carbonyl]-4-oxo-L-ornithine]-4-[4-(4-hydroxyphenyl)-L-threonine]-5-L-serine-echinocandineB.

311 μl of trimethylsilyl iodide is added to a suspension containing 809mg of the product of Stage A and 19 ml of acetonitrile. The reactionmixture is maintained under agitation for 15 minutes at 60° C. and undera nitrogen atmosphere. The mixture is poured into a saturated solutionof sodium thiosulphate followed by evaporation. The residue obtained ischromatographed on silica, eluting with a methylenechloride/methanol/water mixture 86/13/1. The sought product is obtained.Yield 55%.

Stade C:1-[4-[((2S)-2-amino-2-methylethyl)amino]-N2-[[4′-(octyloxy)[1,1′-biphenyl]-4-yl]-carbonyl]-L-ornithine]-4-[4-(4-hydroxyphenyl)-L-threonine]5-L-serine-echinocandineB trifluoroacetate (isomer A and isomer B).

A solution containing 62.5 ma of (S)-(−)diaminopropane dihydrochloride,2.25 ml of methanol, triethylamine in order to obtain a pH of 6, a fewgrains of activated siliporite and 150 mg of the product of the previousstage is agitated for a few minutes at 20° C. 6 mg of NaBH, CN isintroduced. Agitation is carried out for 15 hours at 20° C. and aftersemi-preparative HPLC purification eluent: CH₃CN. H₂OTFA (50-50-0.02)11.5 mg, of isomer A, 13 mg of isomer B are obtained.

EXAMPLE 21-[4-[[(1H-benzimidazol-2-yl)-methyl)-]-amino]-N2-[[4″-(pentyloxy)[1,1′:4′,1″-terphenyl]-4-yl]-carbonyl]-L-ornithine]-4-[4-(4-hydroxyphenyl)-L-threonine]-5-L-serineechinocandin B trifluoroacetate (isomer B).

By operating as previously startin, from the nucleus ofdeoxynmulundecandine prepared in Preparation 1 and obtaining1-[(4R,5R)-4,5-dihydroxy-N2-[[4″-(pentyloxy)[1,1′:4′.1″-terphenyl]-4-yl]carbonyl]-L-threonine]-4-[4-(4-hydroxyphenyl)-L-threonine]-5-L-serineechinocandin B as intermediate product and the corresponding 4-oxoderivative, the sought product was obtained. Isomer A=7.4 mg, isomerB=1.2 mg.

EXAMPLE 3 Trans1-[4-[(2-aminocyclo-hexyl)-amino]-N2-[[4″-(pentyloxy)[1,1′:4′,1″-terphenyl]-4-yl)]-carbonyl]-L-ornithine]-4-[4-(4-hydroxyphenyl)-L-threonine]-5-L-serine-echinocandinB trifluoroacetate (isomer A).

By operating as previously, starting from 166 mg of the 4-oxo derivativeprepared above and 78. mg of (1R, 2R)1-2-diaminocyclohexane, 462 mg ofcrude product is obtained which is chromatographed on silica elutingwith a methylene chloride, methanol, H₂O, acetic acid mixture 86/13/2/1.100 mg of product is obtained which is purified by semi-preparative HPLCagain with a CH₃CN/H₂O/TFA mixture=50/50/0.1. 55 mg of isomer A, 5.2 mgof isomer B are obtained.

EXAMPLE 41-[4-[(2(S)-aminopropyl)-amino]-N2-[[4″-(pentyloxy)[1,1′:4′,1″-terphenyl]-4-yl]-carbonyl]-L-ornithine]-4-[4-(4-hydroxyphenyl)-L-threonine]-5-L-serineechinocandin B trifluoracelate (isomer A).

By operating as previously, the sought product was obtained.

EXAMPLE Pharmaceutical Composition

Tablets were prepared containing:

-   -   Product of Example 3 isomer A . . . 150 mg    -   Excipient s.q.f. . . . 1 g (Detail of excipient: starch, talc,        magnesium stearate).

Pharmacological Study

A—Inhibition of the Glucan Synthase of Candida albicans.

Candida albicans membranes were purified according to the processdescribed by Tang et al Antimicrob. Agents Chemother 35, 99-103, 1991.22.5 μg of membrane proteins are incubated in a mixture of 2 Mm of14C-UDP glucose (specific activity=0.34 mCi./mmol, 50 μg of α-amylase, 1Mm of dithiotreitol (DTT), 1 Mm EDTA, 100 Mm NaF, 7 μM of GTP-γ-S, 1M ofsucrose and 50 Mm of Tris-HCL (pH 7.8) in a volume of 100 μl. The mediumis incubated at 25° C. for 1 hour and the reaction is terminated byadding TCA at a final concentration of 5%. The reaction mixture istransferred onto a pre-humidified glass fibre filter. The filter iswashed, dried and its radioactivity is counted. Mulundocandine is usedas a positive control. Control of the vehicle is carried out with thesame quantity of 1% DMSO. The results obtained show that in this testthe products of the invention show a good activity in particular theproducts of Example 3 isomer A.

B—Activity on the Aspergillus fumigatus Enzyme.

The enzyme is prepared according to the process of Beaulieu etal.(Antimicrob. Agents Chenother 38, 937-944, 1994. The protocol used isidentical to the protocol described above for the enzyme of Candidaalbicans except that dithiotreitol is not used in the reaction-mixture.

In this test the products show a good activity.

1. A compound selected from the group consisting of all possibleisomeric forms and their mixtures, a compound of the formula

either R₁ is hydrogen or methyl and R₂ is selected cyclohexylsubstituted by an amine. R₃ is selected from the group consisting ofhydrogen, methyl and hydroxyl. R₄ is hydrogen or hydroxyl, R is selectedfrom the group consisting of alkyl and cycloalkyl of up to 30 carbonatoms, optionally containing at least one heteroatom, at least oneheterocycle and alkyl or cyclic acyl of up to 30 carbon atoms optionallycontaining at least one heteroatom, andaef at least one heterocycle. Tis selected from the group consisting of hydrogen, methyl, —CH₂CONH₂,—CH₂—C≡N, and —(CH₂)₂NH₂, Y is selected from the group consisting ofhydrogen, hydroxyl, halogen and —OSO₃H or a salt thereof, W is hydrogenor OH, Z is hydrogen or methyl or its non-toxic, pharmaceuticallyacceptable acid addition salt.
 2. The compound of claim 1 in which T ishydrogen.
 3. The compound of claim 1 in which W is hydrogen.
 4. Thecompound of claim 1 in which Z is methyl.
 5. The compound of claim 1 inwhich Y is hydrogen.
 6. The compound of claim 1 in which R₃ is methyl.7. The compound of claim 1 in which R₄ is hydroxyl.
 8. The compound ofclaim 1 in which R is selected from the group consisting of


9. The compound of claim 8 in which R is


10. The compound of claim 8 in which R is


11. The compound of claim 1 in which R₁ is hydrogen.
 12. The compound ofclaim 1 in which R₂ is

13-14. (canceled)
 15. The compound of claim 1 trans1-[4-[(2-aminocyclo-hexyl)-amino]N2-[[4″(pentyloxy)[1,1′:4′,1″-terphenyl]-4-yl-carbonyl]-L-orithine]-4-[4-hydroxyphenyl)-L-threonine]-S-L-serine-echinocandinB trifluoroacetate (isomer A). 16-18. (canceled)
 19. An antifungalcomposition comprising an antifingally effective amount of a compound ofclaim 15 and an inert pharmaceutical carrier.
 20. A method of treatingfungal infections in warm-blooded animals comprising administering towarm-blooded animals in need thereof an antiflngally effective amount ofa compound of claim 15.